Publications having subject matter which relates to the present invention primarily as background are "Optical Measurement of the Thickness of a Film Adsorbed from a Solution", authors Irving Langmuir et al, Journal of the American Chemical Society, Vol. 59 (July-December 1937) page 1406; and "Interactions Among Human Blood Proteins at Interfaces", Leo Vroman et al, Federation Proceedings, Vol. 30, No. 5 (Sept.-Oct. 1971) pages 1494-1502.
Immunological reactions are highly specific interactions in which an antigen combines with an antibody specific to the antigen to form an immunological complex. Immunological reactions taking place within a biological system such as an animal are vital to the animal in combating disease. In a biological system, the entry of a foreign protein, i.e., the antigen, causes the biological system to produce the specific antibody proteins to the antigen in a process not fully understood at this time. The antibody protein molecules have available chemical bonding sites which complement those on the antigen molecule and so the antigen and antibody combine to form an immunologically complexed protein.
Antibodies are produced by biological systems in response to invasion thereof by foreign proteins. Hence, the detection of antibodies present in a biological system is of medical diagnostic value in determining the antigens to which the system has been exposed. Conversely, the detection of certain antigens of a biological system also has medical diagnostic value; examples of diagnostic detection of antigens include detection of HCG protein molecules in urine as a test for pregnancy, and detection of hepatitis associated antigen molecules in blood of prospective blood donors.
In addition to the immunological reaction which occurs between specific protein antigens and specific protein antibodies resulting in the formation of a protein antigen-protein antibody complex, other immunological complexing reactions between immunologically reactive antigens and antibodies are also contemplated by this invention. In addition, specific reactions between other biological particles, such as enzymes and their substrates, are also among the methods contemplated herein and are embraced by the term "immunological reaction" as used herein. Furthermore, the terms "antigen" and "antibody" as used herein are intended to encompass such terms as enzymes, substrates of enzymes, and similar biological particles. As will also be seen, the method is versatile enough to permit substitution of a specific antibody for the corresponding antigen and the antigen for the corresponding specific antibody.
For instance, the following systems include biological particles which are capable of undergoing the immunological reactions described herein:
Viruses PA2 Bacteria and Bacterial toxins PA2 Fungi PA2 Parasites PA2 Animal tissue PA2 Animal body fluids, and the like.
With respect to viruses, the antigens are viral cultures, or parts thereof, and the antibody specific thereto can be produced by administration to a living host. Illustratively, antigen-antibody complexes in the following virus systems are useful in the herein disclosed procedure:
Rubella virus culture (antigen) - Rubella virus antibody; PA1 polio virus culture (antigen) - polio virus antibody; PA1 vesicular stomatitis virus (VSV) culture (antigen) - VSV antibody. PA1 tetanus toxoid suspension (antigen) - tetanus antibody; PA1 diphtheria toxin suspension (antigen) - diphtheria antibody; PA1 Neisseria gonorrhoeal suspension (antigen) - gonorrhea antibody; PA1 Treponema pallidum suspension (antigen) - syphilis antibody. PA1 Aspergillus extract suspension (antigen) - aspergillus fungus antibody; PA1 Candida extract suspension (antigen) - candida fungus antibody. PA1 Trypsin extract - trypsin antibody PA1 chymotrypsin extract - chymotrypsin antibody PA1 pepsin extract - pepsin antibody PA1 ribonuclease extract - ribonuclease antibody PA1 thrombin extract - thrombin antibody PA1 amylase extract - amylase antibody PA1 penicillinase extract - penicillinase antibody PA1 insulin - insulin antibody
Regarding bacteria and bacterial toxins, the antigens are the particular bacteria or bacterial toxin, or parts thereof, and the antibody is produced by injection into a living host. The following are illustrative examples of antigen-antibody pairs which can be used in the present method:
As for fungi, the antigens are antigenic extracts of fungal suspensions and the antibody is the fungal antibody produced by injection into a living host. Antigen-antibody complexes of fungi systems are illustrated by the following:
Antigens and antibodies in parasite systems are obtained in a similar fashion to those of fungi. The system Toxoplasma gondii extract (antigen) - Toxoplasma gondii antibody is a typical example.
By the term polysaccharides is meant a system wherein the antigen is a carbohydrate antigen. An example of such an antigen-antibody containing system is pneumococcus polysaccharides (antigen) - Pneumococcus antibody.
In addition to the typical enzyme - enxyme substrate reaction which is intended to be covered herein, enzymes themselves, or parts thereof may be utilized as antigens and the antibody is the particular enzyme antibody elaborated by a living host after injection. Illustrative antigen-antibody complexes of enzyme systems are:
With respect to hormones, the antigenic constituent is usually found in a hormone extract and the antibody is the particular hormone antibody elaborated by the living organism after injection. An exemplary antigen-antibody complex is:
As presently practiced, both the collection and purification and the diagnostic utilization of immunologically reactive antigens and antibodies rely upon the complexing of the antigen and antibody specific to the antigen. The classic example of these diagnostic uses is the blood typing procedure in which blood samples are mixed with A and B type serum antibodies and blood type is determined by observing any agglutination occurring in the blood samples. The human chorionic gonadotrophin protein pregnancy test as currently practiced is an inhibition test. The test is performed by mixing a quantity of HCG anti-serum into a urine specimen. A plurality of polystyrene spheres which have been coated with HCG protein are then introduced into the previously prepared urine specimen. The polystyrene spheres will agglutinate if, but only if, HCG protein is absent from the urine specimen. If HCG protein is absent from a urine specimen, the HCG protein on the polystyrene spheres complexes with the HCG anti-serum previously introduced in the urine specimen and the spheres agglutinate. If, on the other hand, HCG protein is present in the urine specimen in sufficient quantity, it complexes with the previously introduced HCG anti-serum forming a complex which precipitates out of the specimen so that the previously introduced anti-serum is no longer available to complex with the HCG protein on the spheres to cause agglutination thereof. The present HCG protein pregnancy test could be simplified by adhering HCG anti-serum onto the polystyrene spheres and directly testing a urine specimen. In this case, the polystyrene spheres would agglutinate if, but only if, HCG protein is present in the specimen.
It appears that the reason this simpler procedure has not been employed is that the available HCG anti-sera are complex mixtures containing a large proportion of constituents other than HCG antibodies. The additional effort required in the prior art to extract the antibodies from the HCG anti-sera made the inhibition test, utilizing sera directly, preferable in the prior art. However, in accordance with one embodiment of this invention, a procedure is provided whereby HCG antibodies are efficiently separated from sera and, which procedure, furthermore, produces diagnostic apparatus whereby the simpler, direct test is performable.
The present procedure for obtaining purified concentrations of antibodies comprises the steps of stimulating the production of antibodies in an animal by introducing the antigen into the animal's system, obtaining blood serum from the animal which contains the antibodies in a dilute form, and mixing a quantity of the specific antigen into the serum. The mixture of antigen and antibody complexes and precipitates out of the serum solution. The remaining constituents of the serum are drawn off and the antibody-antigen precipitate is dissolved in an acid which severs the complexing bonds. At this point one has a solution of antigen and antibody molecules in acid. Since the antibody and antigen molecules have differing physical characteristics, for example, weight, they may be separated from each other by mechanical means, for example, by centrifuging.
It is known that the antibody-antigen complexing reaction will take place when an antigen is adsorbed at a surface. The complexing reaction at a surface has been observed by means of an ellipsometer.
A principal object of this invention is to provide method and apparatus for concentrating and purifying antigens and antibodies by means of controlled immunological reactions occurring at a surface.